Phencyclidine and phencyclidine metabolites assay, tracers, immunogens and antibodies

ABSTRACT

The present invention is directed to a fluorescence polarization assay for phencyclidine and phencyclidine derivatives, to the various components needed for preparing and carrying out such an assay, and to methods of making these components. Specifically, tracers, immunogens and antibodies are disclosed, as well as methods for making them. The tracers and the immunogens are made from substituted phencyclidine compounds. A fluorescein moiety is included in the tracer, while a poly(amino acid) forms a part of the immunogen. The assay is conducted by measuring the degree of polarization retention of plane polarized light that has been passed through a sample containing antiserum and tracer.

This is a division of co-pending application Ser. No. 866,193, filed onMay 21, 1986, now abandoned. On Jun. 7, 1990, a continuation-in-partapplication of co-pending application Ser. No. 866,193 was filed withthe U.S. Patent and Trademark Office.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a method and reagents for performing afluorescence polarization immunoassay (FPIA) to determine the presenceor amount of phencyclidine and phencyclidine metabolites in fluids,especially biological fluids such as urine, serum or plasma, and to amethod of making the reagents. The invention relates particularly to (1)reagents (tracers and antibodies) for determining the presence or amountof phencyclidine and phencyclidine metabolites in a sample; (2)immunogen compounds used to raise antibodies; (3) synthetic methods (formaking tracer and immunogen compounds); and (4) analytical methods forconducting the assay.

2. Background Art

Phencyclidine is a synthetic drug with potent analgesic and anestheticproperties. The drug has been shown to produce serious and prolongedpost-anesthetic confusion and delirium. Its tendency to producehallucinations, euphoria, distortions in perceptions, and feelings ofdissociation have lead to illicit use and abuse. Recurring abuse hasintensified efforts to prevent its manufacture and distribution.Consistent with these efforts, there exists a need for detection methodsthat are rapid, reliable and selective for phencyclidine andphencyclidine metabolites.

Phencyclidine is metabolized into two major metabolites,4-phenyl-4-piperidinocyclohexanol and1-(1-phenylcyclohexyl)-4-hydroxypiperidine, which are excreted mostly inthe urine along with the corresponding glucuronide conjugates. Detectionof either phencyclidine or phencyclidine metabolites indicatesphencyclidine use.

The biological fluid most frequently tested is urine. Urine samples arenon-invasive of the body, and are generally more accessible than bloodsamples. Although testing of other biological fluids is a possibility,they have not been extensively investigated with respect to such assays.

In the past, urine samples have been tested for the presence ofphencyclidine and phencyclidine metabolites by thin layer chromatography(TLC), enzyme immunoassay (EIA), gas chromatography (GC) or highperformance liquid chromatography (HPLC) assays. These methods are notwithout drawbacks; e.g., the assay time involved in these methods istypically lengthy.

In assays for other substances, competitive binding immunoassays haveprovided a more satisfactory alternative. Typically, competitive bindingimmunoassays are used for measuring ligands in a test sample. (Forpurposes of this disclosure, a "ligand" is a substance of biologicalinterest to be quantitatively determined by a competitive bindingimmunoassay technique). The ligands compete with a labeled reagent (a"ligand analog" or "tracer") for a limited number of receptor bindingsites on antibodies specific to the ligand and ligand analog. Theconcentration of ligand in the sample determines the amount of ligandanalog which binds to the antibody; the amount of ligand analog thatwill bind is inversely proportional to the concentration of ligand inthe sample, because the ligand and the ligand analog each bind to theantibody in proportion to their respective concentrations.

FPIA techniques provide a quantitative means for measuring the amount oftracer-antibody conjugate produced in a competitive binding immunoassay.Such procedures are based on the principle that a fluorescent labeledcompound, when excited by plane polarized light, will emit fluorescencehaving a degree of polarization inversely related to its rate ofrotation. Accordingly, when a tracer-antibody conjugate having afluorescent label is excited with plane polarized light, the emittedlight remains highly polarized because the fluorophore is constrainedfrom rotating between the time that light is absorbed and emitted. Incontrast, when an unbound tracer is excited by plane polarized light,its rotation is much faster than the corresponding tracer-antibodyconjugate and the molecules are more randomly oriented. As a result, thelight emitted from the unbound tracer molecules is depolarized.

A problem that heretofore has prevented the accurate determination ofphencyclidine and other "drugs of abuse" in urine by FPIA techniques isthat of riboflavin interference. Riboflavin, or vitamin B₂, is a commonconstituent of many foods and of commercially available vitaminsupplements. Riboflavin is excreted primarily in the urine and has afluorescence spectrum quite similar to that of fluorescein. As a result,the presence of riboflavin in even moderate amounts in urine samplescreates an interference which can produce erroneous data. While ordinaryconsumption of riboflavin is unlikely to produce more than trace amountsof riboflavin in the urine, test results can readily be distorted by theconsumption of excessive quantities of vitamin supplements by personswishing to prevent detection of phencyclidine.

The present invention offers an advance in the art in that highlysensitive tracers, a method for making the tracers, and an assay usingthe tracers are provided specifically for the determination ofphencyclidines and phencyclidine metabolites without riboflavininterference.

SUMMARY OF THE INVENTION

The present invention is directed to a fluorescence polarization assayfor phencyclidine and phencyclidine metabolites; to tracers, immunogensand antibodies for use in the assay; and to methods for making thetracers, immunogens and antibodies.

A first aspect of the invention relates to the discovery of uniquetracers and immunogens having novel structures. According to the firstaspect of the invention, the tracers and the immunogens can both berepresented by the structural formula shown in FIG. 5 where:

W is CH or N;

R is a linking group including up to 4 heteroatoms and having a total offrom 0 to 8 carbon atoms and heteroatoms;

Z is NH, CO or CNH;

n is 0 or 1 when W is N and n is 1 when W is CH; and

Q is a poly(amino acid), a poly(amino acid) derivative, fluorescein or afluorescein derivative.

When Q is a poly(amino acid) or a derivative thereof, the compound canbe used as an immunogen. When Q is fluorescein or a derivative thereof,the compound can be used as a tracer.

A second aspect of the invention relates to antibodies raised by thenovel immunogens of the invention. According to the second aspect of theinvention, antibodies are prepared in response to a compound accordingto the aforementioned structural formula (FIG. 5), when Q is apoly(amino acid) or a derivative thereof.

According to a third aspect of the invention, an immunogen is made by amethod comprising the step of coupling a compound represented by thestructural formula shown in FIG. 2, where:

W is CH or N;

R is a linking group including up to 4 heteroatoms and having a total offrom 0 to 8 carbon atoms and heteroatoms when Z is NH, CN or OH andhaving a total of from 1 to 8 carbon atoms when Z is COOH or CHO;

Z is NH₂, COOH, CN, CHO or OH; and

n is 1 when W is CH and n is 0 or 1 when W is N; with a poly(amino acid)or a derivative of a poly(amino acid).

According to a fourth aspect of the invention, a method is provided formaking a tracer by coupling a compound represented by the structuralformula shown in FIG. 3, where:

W is CH or N;

R is a linking group including up to 4 heteroatoms and having a total offrom 0 to 8 carbon atoms and heteroatoms when Z is NH, CN or OH andhaving a total of from 1 to 8 carbon atoms when Z is COOH or CHO;

Z is NH₂, COOH, CN, CHO or OH; and n is 1 when W is CH and n is 0 or 1when W is N; with fluorescein or a derivative of fluorescein.

A fifth aspect of the invention relates to the elimination of potentialfluorescence interference by riboflavin. Riboflavin binding protein(RBP) is added either directly to each sample or to one or more of thereagents utilized in the assay, wherein it binds all riboflavin presentinto RBP-riboflavin complexes, thus eliminating fluorescenceinterference.

According to a sixth aspect of the invention, a process for detecting ormeasuring the concentration of phencyclidine and phencyclidinemetabolites is provided. A sample is contacted with phencyclidinederivative antiserum, and a fluorescein-containing phencyclidinederivative capable of producing a detectable fluorescence polarizationresponse to the presence of the phencyclidine derivative antiserum.Plane polarized light is then passed through the solution to obtain afluorescence polarization response, and this response is detected as ameasure of the amount of phencyclidine and phencyclidine metabolite inthe sample.

Further objects and attendant advantages of the invention will be bestunderstood from a reading of the following detailed description takentogether with the Examples and appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawing Figures hereof the symbol "F1" represents fluorescein ora fluorescein derivative and the various other symbols are noted in theDetailed Description, infra.

FIG. 1 shows the general structure of the class of phencyclidine to tbequantitatively or qualitatively determined in accordance with thepresent invention.

FIG. 2 shows a class of reactants for a method of making a tracer inaccordance with the present invention.

FIG. 3 shows a class of reactants for a method of making an immunogen inaccordance with the present invention.

FIG. 4 shows the alternate structural formula and names of thefluorescein moiety included in the tracers of the present invention.

FIG. 5 shows a general structural formula for the tracers and theimmunogens of the present invention.

FIG. 6 shows a general structural formula for the immunogens of thepresent invention.

FIG. 7 shows a general structural formula for the tracers of the presentinvention.

FIG. 8 shows a structural formula for preferred immunogens of thepresent invention.

FIG. 9 shows a structural formula for preferred tracers of the presentinvention.

FIG. 10 shows a precursor for the immunogens shown in FIGS. 6 and 8 andfor the tracers shown in FIGS. 7 and 9.

FIGS. 11-1 through 11-10 show various linkages that couple thefluorescein moiety to the precursor at the Z position in FIG. 10, whenFIG. 10 represents a precursor for the tracers shown in FIGS. 7 and 9.

FIGS. 12 through 16 show various examples of structures of tracers inaccordance with the present invention.

FIGS. 17 through 19 show various examples of structures of haptenreactants used to form the immunogens of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The various aspects of the invention will now be discussed in relationto the Figures and/or the Examples.

The present invention involves the use of fluorescein and derivatives offluorescein. In particular, a necessary property of fluorescein and itsderivatives for the usefulness of the tracer compounds of the presentinvention is the fluorescence of fluorescein. Fluorescein exists in twotautomeric forms, illustrated in FIG. 4, depending on the acidconcentration (pH) of the environment. In the open (acid form), thereare a number of conjugated double bonds which make that form offluorescein (and compounds containing a fluorescein moiety) capable ofabsorbing blue light and emitting green fluorescence after an excitedstate lifetime of about 4 nanoseconds. When the open and closed formscoexist, the relative concentration of molecules in the open and closedforms is easily altered by adjustment of the pH level. Generally, thetracer compounds of the present invention exist in solution asbiologically acceptable salts such as sodium, potassium, ammonium andthe like, which allows the compounds to exist in the open, fluorescentform, when employed in the analytical methods of the present invention.The specific salt present will depend on the buffer employed to adjustthe pH level. For example, in the presence of a sodium phosphate buffer,the compounds of the present invention will generally exist in the openform, as a sodium salt.

As used herein, the term "fluorescein," either as an individual compoundor as a component of a larger compound, is meant to include both theopen and closed forms, if they exist for a particular molecule, exceptin the context of fluorescence. An open form is necessary for thefluorescence to occur.

The numbering of carbon atoms of the fluorescein molecule varies,depending upon whether the open or closed form of the molecule isconsidered. Accordingly, the literature concerning fluorescein and itscompounds is not uniform as to carbon atom numbering. In the closedform, the para-carbon to the carbonyl of the lactone on the phenyl ringis numbered 6 (this is sometimes denominated "isomer II"). In the openform, the para-carbon to the carboxylic acid group on the phenyl ring isnumbered 5 (this is sometimes denominated "isomer I"). FIG. 4illustrates these isomers. For the purpose of this disclosure thenumbering of the closed form is adopted because the raw materials usedin the syntheses are most popularly numbered with that system. Thecarbon atom of fluorescein and its compounds which is opposite thecarboxyl group is therefore numbered "6" for the purposes of the presentdisclosure.

A tracer which is not complexed to an antibody is free to rotate in lessthan the time required for absorption and re-emission of fluorescentlight. As a result, the re-emitted light is relatively randomly orientedso that the fluorescence polarization of a tracer not complexed to anantibody is low, approaching zero. Upon complexing with a specificantibody, the tracer-antibody complex thus formed assumes the rotationof the antibody molecule which is slower than that of the relativelysmall tracer molecule, thereby increasing the polarization observed.Therefore, when a ligand competes with the tracer for antibody sites,the observed polarization of fluorescence of the tracer-antibody complexbecomes a value somewhere between that of the tracer and tracer-antibodycomplex. If a sample contains a high concentration of the ligand, theobserved polarization value is closer to that of the free tracer, i.e.,low. If the test sample contains a low concentration of the ligand, thepolarization value is closer to that of the bound tracer, i.e., high. Bysequentially exciting the reaction mixture of an immunoassay withvertically and then horizontally polarized light and and analyzing onlythe vertical component of the emitted light, the polarization offluorescence in the reaction mixture may be accurately determined. Theprecise relationship between polarization and concentration of theligand to be determined is established by measuring the polarizationvalues of calibrators with known concentrations. The concentration ofthe ligand can be extrapolated from a standard curve prepared in thismanner.

The particular tracers formed in accordance with this invention havebeen found to produce surprisingly good assays, as will be demonstratedlater in this disclosure.

The Reagents

Both the immunogens and the tracers of the present invention can berepresented by the general structural formula set forth in the Summaryof the Invention, and illustrated in FIG. 5. When Q is a poly(aminoacid), the structure represents the immunogen; when Q is a fluoresceinderivative, the structure represents the tracer.

The objective is to have competition between phencyclidine andphencyclidine metabolites and the tracer for the recognition sites ofthe antibody. Great variations in the structure of the haptens andtracers are allowed in achieving this goal. For the purposes of thisinvention, "haptens" are precursors of the immunogens, comprisinggenerally a substituted phencyclidine derivative and a linking group tothe poly(amino acid) carrier.

The Structure of the Immunogens

Usable antibodies can be produced from a variety of phencyclidinederivatives. Such antibodies are useful in phencyclidine andphencyclidine metabolites assay according to the invention when combinedwith the appropriate tracer.

The immunogens of the present invention have the general structuralformula shown in FIG. 6, and in the preferred form of the invention, theimmunogens have the structural formula shown in FIG. 8. This structureis preferred because the best recognition of the common moiety ofphencyclidine and phencyclidine metabolites, the phenyl ring, occurswhen the piperidine ring is substituted at a position as distant aspossible from the phenyl ring. Although bovine serum albumin is thepoly(amino acid) in this preferred form, it should be understood thatvarious protein carriers may be employed, including albumins, serumproteins, e.g., globulins, ocular lens proteins, lipoproteins and thelike. Illustrative protein carriers include, in addition to bovine serumalbumin, keyhole limpet hemocyanin, egg ovalbumin, bovinegamma-globulin, thyroxine binding globulin, etc. Alternatively,synthetic poly(amino acids) may be prepared having a sufficient numberof available amino groups such as lysines. The correspondingglutaraldehyde derivative of the above poly(amino acid) carriers mayalso be employed when the hapten coupling group is an amino group.

The immunogens can be prepared by coupling a compound of the class shownin FIG. 2 with a poly(amino acid) or a derivative of a poly(amino acid),as will be discussed in the context of the synthetic method and theExamples below.

The Structure of the Tracers

The possible variations in the structure of the tracers of the inventionare even greater than the possible variations in the structure of thehaptens thereof. The tracers of the present invention have the generalstructural formula shown in FIG. 7, where F1 represents a fluoresceinmoiety or a fluorescein derivative. In a preferred form of theinvention, the tracers have the structural formula shown in FIG. 9.

The tracer is a phencyclidine derivative that is linked to a fluoresceinderivative by, e.g., an amido, amidino, triazinylamino, carbamido,thiocarbamido, carbamoyl, thiocarbamoyl, or sulfomylcarbamoyl group, asshown in FIG. 11. The tracers are prepared by linking the appropriatefluorescein derivative to a phencyclidine derivative containing anamino, carboxylic acid, hydroxy, imidate, hydrazide, chloroformate,chlorothioformate, chlorosulfonyl-carbamoyl, isocyanate, thioisocyanate,or similar group, as will be discussed in the context of the syntheticmethod and the Examples below.

By way of example, any of the following fluorescein derivatives can beused:

    ______________________________________                                        FlNH.sub.2     fluorescein amine                                              FlCO.sub.2 H   carboxyfluorescein                                             FlNHCOCH.sub.2 I                                                                             α-iodoacetamidofluorescein                                ##STR1##      2,4-dichloro-1,3,5,-triazin-2-yl amino-fluorescein (DTAF)       ##STR2##      4-chloro-6-methoxy-1,3,5-triazin-2- ylamino fluorescein        FlNCS          fluorescein thioisocyanate                                     ______________________________________                                    

The Antibodies

The antibodies of the present invention are prepared by developing aresponse in animals to the immunogens described above. The immunogen isadministered to animals such as rabbits or sheep by a series ofinjections, in a manner well-known to those skilled in the art.

Synthetic Methods

Both the immunogens and the tracers of the present invention can be madefrom a precursor having the general structural formula shown in FIG. 10,where:

W is CH or N;

R is a linking group including up to 4 heteroatoms and having a total offrom 0 to 8 carbon atoms and heteroatoms when Z is NH₂, CN or OH andhaving a total of from 1 to 8 carbon atoms and heteroatoms when Z isCOOH or CHO;

Z is NH₂, COOH, CN, CHO, or OH when the preparation is directed to animmunogen, and Z is NH₂, COOH, CN or OH when the preparation is directedto a tracer; and

n is 1 when W is CH and n is 0 or 1 when W is N.

The Synthesis of the Immunogens

The immunogens of the present invention are made by coupling a hapten,such as that shown by the general structure of FIG. 2 when Z is NH₂,COOH, CN, CHO, or OH, to a poly(amino acid). The poly(amino acid) moietycan be linked to the hapten by an amide, an amidine, an alkyl, a urea, athiourea, a carbamate, or a thiocarbamate linkage. In a preferredembodiment, the poly(amino acid) is bovine serum albumin (BSA) and thehapten is shown in FIG. 18. The hapten is preferably coupled underconditions normally used to form carbamate linkages, which conditionsare well known to those skilled in the art. It is most preferred that pHconditions approximating pH 8.0 be used for forming the desiredcarbamate linkages, as these are the most effective for forming theselinkages in this context.

The immunogens are prepared by coupling a hapten containing an --NH₂,--CO₂ H, --CONHNH₂, --CNOR, --CHO, --NCO, --NCS, --OCOCl or --OCSClgroup to a poly(amino acid). The --NH₂ case can be coupled by activatingthe carboxylic acid group on the poly(amino acid) in the presence of the--NH₂ group. The activation of the carboxylic acid groups on thepoly(amino acid) can be accomplished by mixing the hapten and thepoly(amino acid) with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC), N,N'-dicyclohexylcarbodiimide (DCC),1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate,or the like. The --CO₂ H case is also coupled by the activation method(EDC) or the active ester method. The --CONHNH₂ case is coupled in thesame manner as for the non-aromatic amino case. The --CNOR case which isprepared from the corresponding cyano compound, is coupled directly tothe poly(amino acid). The --CHO case is coupled to the poly(amino acid)by reductive amination. The poly(amino acid) is mixed with the --CHOhapten and the resulting imine is reduced with sodium cyanoborohydrideto yield alkylated amines on the poly(amino acid). The isocyanate(--NCO) and isothiocyanate (--NSC) cases, which are prepared from thecorresponding amino compound and chloroformate (--OCOCl) andchlorothioformate (--OCSCl) cases which are prepared from thecorresponding alcohol compound, produce urea, thiourea, carbamate andthiocarbamate linkages, respectively. This is accomplished by directcoupling of the hapten to the poly(amino acid).

The synthesis of the above haptens (immunogen precursors) areaccomplished in very similar ways. FIG. 2 shows an immunogen precursorclass in accordance with a preferred embodiment of the method of thepresent invention.

In general, the hapten is prepared by reaction of the appropriatepiperidine derivative with cyclohexanone in the presence of cyanide. Thecoupled product is then reacted with phenyl magnesium bromide to yieldthe hapten precursor. The hapten precursor is then converted into thehapten. ##STR3##

In the case where W is N, X is benzyl amine. The benzyl group is removedafter formation of the phencyclidine derivative. This secondary amine isa suitable hapten. It is possible to alkylate the amine with an alkylhalide (Cl, Br or I), e.g., bromoacetonitrile, 3-bromopropanol,4-bromobutyric acid or the like, to prepare suitable haptens. It is alsopossible to form the chloroformamide derivative, which could make asuitable hapten, or form an amide derivative with an active ester, e.g.,succinic anhydride, cyanoacetyl chloride or the like, containing asuitable group useful for coupling to a carrier protein. In the casewhere W is CH, X is CHOH, after formation of the phencyclidinederivative, a large variety of haptens may be prepared. The alcohol maybe alkylated with an alkyl halide (Cl, Br, or I), containing a suitablegroup useful for coupling to a carrier protein, by standard proceduresto form ether derivatives. The alcohol may be converted into thecorresponding halogen derivative, such as, bromo, chloro or iodo, whichwill react with carbanion, alcohol or amine derivatives of compoundscontaining a suitable group for coupling to a carrier protein. Thealcohol can be oxidized to the corresponding ketone which may bederivatized by known methods to a variety of compounds containing asuitable group useful for coupling to a carrier protein, e.g., Wittingreagents, alkoxyamine compounds, reductive amination with aminocompounds or the like. Reductive amination of the ketone with ammoniumacetate results in the amino derivative where X is CHNH₂. The aminocompound is suitable as a hapten or may be derivatized by known methodsto a variety of hapten compounds analogous to the case where W is N andn is O.

Nitrile derivatives (Z═CN) are converted to alkoxy imidates (Z═CNOR) bytreating the nitrile with anhydrous alcohol and hydrogen chloride gas.The hydrazide derivatives (Z═CONHNH₂) are prepared from thecorresponding carboxylic acid derivatives by active ester coupling withhydrazine or by reacting hydrazine with the corresponding carboxylicester derivative. Amines (Z═NH₂) are convertible to the isocyanate orthioisocyanate derivatives and alcohols (Z═OH) are convertible tochloroformate and chlorothioformate derivatives by reaction of the amineor the alcohol with phosgene or thiophosgene.

Aldehydes and ketones can be condensed with(aminohydroxy)alkylcarboxylic acids, such as NH₂ OCH₂ CO₂ H, to producesubstituted oxime derivatives. The oxime alkyl carboxylic acidderivatives can be partially reduced to the corresponding(aminohydroxy)alkylcarboxylic acid derivatives. The same type ofcondensation and reduction can be accomplished with hydrazine andhydrazine derivatives.

The Synthesis of the Tracers

The tracers of the present invention are made by coupling a fluoresceinmoiety, or a derivative of fluorescein, to the general structure shownin FIG. 10 when Z is NH₂, COOH, CNOR or OH. The fluorescein moiety canbe linked to the amino, carboxyl, imidate or alkoxy functional group byan amide, an amidine, a urea, a thiourea, a carbamate, a thiocarbonate,triazinylamino or sulfonylcarbamate linkage, as shown in FIG. 11. In thepresently preferred embodiment, the fluorescein derivative is6-((4,6-dichloro-1,3,5-triazin-2-yl)amino) fluorescein, and this iscoupled to the precursor 4-amino-1-(1-phenylcyclohexyl)-piperidine in aco-solvent, e.g., methanol, dimethylsulfoxide, or the like, in thepresence of a base, e.g., triethylamine or the like. The structure isshown in FIG. 13. Useable tracers can be prepared from a variety ofphencyclidine derivatives.

All phencyclidine derivatives that have a terminal amino group, such asamino, hydrazinyl, hydrazido or the like, are coupled tocarboxyfluorescein by the active ester method or the mixed anhydridemethod, and coupled to fluorescein isothiocyanate, DTAF or alkoxy DTAFby simply mixing the two materials in solution. The amino group can beconverted to the isocyanate and thioisocyanate groups by reaction withphosgene and thiophosgene, respectively. These are then condensed withaminofluorescein to produce the tracer.

All phencyclidine derivatives that have a terminal carboxylic acidgroup, such as carboxylic acid, (aminohydroxy)alkylcarboxylic acid orthe like, are coupled to aminofluorescein by the active ester method.

All phencyclidine derivatives that have a terminal hydroxy group can becoupled to fluorescein by reaction with DTAF, iodoacetamidofluoresceinor fluorescein isothiocyanate in solution. The hydroxy group can beconverted to the chlorosulfonylcarbamoyl, chloroformate andchlorothioformate groups by reaction with chlorosulfonylisocyanate,phosgene and thiophosgene, respectively. These derivatives are thencoupled to aminofluorescein in solution to produce the tracer.

All phencyclidine derivatives that have a terminal nitrile group areconverted to imidates in anhydrous alcohol in the presence of hydrogenchloride gas. The imidate is then coupled to fluorescein amine insolution to prepare the tracer.

The preparation of the various amino, carboxylic acid, hydroxy andnitrile derivatives of the anilide derivatives were described above inthe immunogen preparation section.

The Assay

The particular tracers and antibodies of the present invention have beenfound to produce surprisingly good results in fluorescence polarizationassays for phencyclidine and phencyclidine metabolites. FIG. 1 shows thegeneral structure of the phencyclidine and phencyclidine metabolitesthat can be quantitatively or qualitatively determined in accordancewith the present invention. The assay of the present invention providesa more rapid phencyclidine and phencyclidine metabolite assay methodthan most prior art methods, because it requires no specimen treatmentbefore analysis. The assay system accurately measures the presence orquantity of phencyclidine and phencyclidine metabolites in a sample,because antibody specificity precludes detection of compounds other thanphencyclidine-like compounds.

In accordance with the analytical methods of the present invention;i.e., the methods of determining phencyclidine and phencyclidinemetabolites by a fluorescence immunoassay procedure using the tracercompounds and immunogens of the invention, a sample containing orsuspected of containing phencyclidine and phencyclidine metabolites isintermixed with a biologically acceptable salt of a tracer and anantibody specific to phencyclidine and phencyclidine metabolites and thetracer. The antibody is produced using the immunogen as described above.The phencyclidine and phencyclidine metabolites and tracer compete forlimited antibody sites, resulting in the formation of complexes. Bymaintaining constant the concentration of tracer and antibody, the ratioof phencyclidine and phencyclidine metabolites-antibody complex totracer-antibody complex that is formed is directly proportional to theamount of phencyclidine and phencyclidine metabolites in the sample.Therefore, upon exciting the mixture with linearly polarized light andmeasuring the polarization of the fluorescence emitted by a tracer and atracer-antibody complex, one is able to quantitatively determine theamount or qualitatively determine the presence of phencyclidine andphencyclidine metabolites in the sample.

The results can be quantified in terms of net millipolarization unitsand span (in millipolarization units). The measurement of netmillipolarization units indicates the maximum polarization when amaximum amount of the tracer is bound to the antibody, in the absence ofany phencyclidine or phencyclidine metabolites. The higher the netmillipolarization units, the better the binding of the tracer to theantibody. The span is an indication of the difference between the netmillipolarization and the amount of tracer bound to the antibody at theminimum phencyclidine concentration above which the sample is defined ascontaining phencyclidine and/or phencyclidine metabolites. A larger spanprovides for a better numerical analysis of data. The preferredantibody-tracer combination has a span of at least 18 millipolarizationunits, but a span of at least 5 millipolarization units is acceptable.It is important to note that the span varies depending on the samplesize used which in turn may alter the preferred combination.

Table I shows the results obtained with various embodiments of thepresent invention, in terms of span and net millipolarization units at asample size of 2 μl. In all instances, bovine serum albumin was used asthe protein carrier. As seen from the data in Table I, an assay producedfrom an immunogen made from the hapten of FIG. 18 used in combinationwith the tracer of FIG. 13 and a 2 μl sample size provides excellentresults. Accordingly, this combination is presently the most preferredform of the invention for a sample size of 2 μl. In addition, thehapten/tracer combinations represented by the combinations of FIGS. 18and 12, FIGS. 18 and 14, and FIGS. 18 and 15, and FIGS. 18 and 16 alsoproduced acceptable results and are alternative preferred combinations.

                  TABLE I                                                         ______________________________________                                        Hapten used                                                                   in Immunogen           Net                                                    for Antibody Tracer    Polarization*                                                                            Span**                                      ______________________________________                                        FIG. 18      FIG. 12   176        16                                          FIG. 18      FIG. 13   172        18                                          FIG. 18      FIG. 14   164        16                                          FIG. 18      FIG. 15   159        15                                          FIG. 18      FIG. 16   187        12                                          FIG. 17      FIG. 12   129        5                                           FIG. 17      FIG. 13   125        5                                           FIG. 17      FIG. 14   102        4                                           FIG. 17      FIG. 15   110        6                                           FIG. 17      FIG. 16   119        4                                           FIG. 19      FIG. 12   137        4                                           FIG. 19      FIG. 13   142        2                                           FIG. 19      FIG. 14   155        6                                           FIG. 19      FIG. 15   140        5                                           FIG. 19      FIG. 16   141        4                                           ______________________________________                                         *In millipolarization units                                                   **In millipolarization units at a phencyclidine concentration of 75 ng/ml     and a 2 μl sample size.                                               

The pH at which the method of the present invention is practiced must besufficient to allow the fluorescein moiety of the tracers to exist intheir open form. The pH may range from about 3 to 12, more usually inthe range of from about 5 to 10, most preferably from about 6 to 9.Various buffers may be used to achieve and maintain the pH during theassay procedure. Representative buffers include borate, phosphate,carbonate, tris, barbital and the like. The particular buffer employedis not critical to the present invention, but the tris and phosphatebuffers are preferred. The cation portion of the buffer will generallydetermine the cation portion of the tracer salt in solution.

Riboflavin binding protein (RBP) is added to the sample or to one ormore of the assay reagents in order to bind any riboflavin present inthe sample into RBP-riboflavin complexes, thus eliminating potentialfluorescence interference. RPB is a protein of approximately 32,000 M.W.which is isolated from egg whites. Upon isolation from the egg, eachmolecule of RBP contains one molecule of riboflavin. This, theholoprotein form of RBP, must be converted to the apoprotein form bydialysis, under acidic conditions, to remove the bound riboflavin. TheRBP apoprotein utilized in the present invention is commerciallyavailable from Sigma Chemical Company, St. Louis, Mo. The amount used isnot critical, provided a sufficient quantity is used to bind all freeriboflavin in the sample.

The preferred method of the improved assay of the present invention willnow be discussed in detail. The assay is a "homogeneous assay," whichmeans that the end polarization readings are taken from a solution inwhich bound tracer is not separated from unbound tracer. This is adistinct advantage over heterogeneous immunoassay procedures such asthose where the bound tracer must be separated from the unbound tracerbefore a reading can be taken.

The reagents for the fluorescence polarization assay of the presentinvention comprise antibody for phencyclidine and phencyclidinemetabolites and tracer. Additionally, largely conventional solutionsincluding a pretreatment solution, a dilution buffer, phencyclidinecalibrators and phencyclidine controls are desirably prepared. Typicalsolutions of these reagents, some of which are described below, arecommercially available in assay "kits" from Abbott Laboratories, AbbottPark, Ill.

All percentages expressed herein are weight/volume unless otherwiseindicated. The tracer formulation presently preferred is 164 nanomolartracer in: 0.1 molar tris buffer at pH 7.9; 10% sodium cholate; 0.1%sodium azide; and 0.01% bovine gamma-globulin. The antiserum formulationcomprises rabbit serum diluted with: 0.1 molar sodium phosphate bufferat pH 7.5; 0.1% sodium azide; 0.01% bovine gamma-globulin; and 2%ethylene glycol (volume/volume). The dilution buffer comprises: 0.1molar sodium phosphate at pH 7.5; 0.1% sodium azide; and 0.01% bovinegamma-globulin. The pretreatment solution comprises: 0.01% bovinegamma-globulin; 0.1 molar tris buffer at pH 7.5; 0.1% sodium azide; and5 ml/ml riboflavin binding protein. Phencyclidine calibrators comprisingphencyclidine in normal human urine at concentrations of 0.0, 25.0,60.0, 120.0, 250.0 and 500.0 nanograms per milliliter, with 0.1% sodiumazide as a preservative are useful. Phencyclidine controls comprisingphencyclidine in normal human urine are provided at concentrations of35.0 and 250.0 nanograms per milliliter with 0.1% sodium azide as apreservative are also useful.

The preferred procedure is especially designed to be used in conjunctionwith the Abbott TDx^(R) Analyzer available from Abbott Laboratories,Irving, Tex. Fifty microliters of urine is required. The calibrators,controls, or unknown samples are pipetted directly into the sample wellof the TDx^(R) sample cartridge. One of the advantages of this procedureis that the sample does not require any special preparation. The assayprocedure from this point is fully automated.

If a manual assay is being performed, then the sample is mixed with thepretreatment solution in dilution buffer and a background reading istaken. The tracer is then mixed with the assay. The antibody is thenfinally mixed into the test solution. After incubation, a fluorescencepolarization reading is taken.

The fluorescence polarization value of each calibrator, control orsample is determined and is printed on the output tape of an instrumentsuch as the Abbott TDx^(R) Analyzer. A standard curve is generated inthe instrument by plotting the polarization of each calibrator versusits concentration using a nonlinear regression analysis. Theconcentration of each control or sample is read off the storedcalibration curve and printed on the output tape.

With respect to the foregoing preferred procedure, it should be notedthat the tracer, antibody, pretreatment solution, calibrators andcontrols should be stored between about 2° C. and about 8° C. while thedilution buffer should be stored at ambient temperature. A standardcurve and controls should be run every two weeks, with each calibratorand control run in duplicate. All samples can be run in duplicate.

It should be understood that the foregoing detailed description and thefollowing Examples are intended to be illustrative, but not limiting,with respect to the scope of the present invention. Variousmodifications will become apparent to one skilled in the art, and thusit is intended that the scope of the invention be defined solely by theclaims and legal equivalents thereof.

EXAMPLES

Examples I through XVI describe experiments that were performed inaccordance with the concepts of the present invention. Examples Ithrough III are directed to preparation of an immunogen useful forproducing antibody; Examples IV through IX and XI and XII are directedto the synthesis of precursors for immunogens and tracers; and ExamplesX and XIII through XVI are directed to the preparation of tracers.

EXAMPLE I 1-(1-Phenylcyclohexyl)piperazine Immunogen

1-(1-Phenylcyclohexyl)piperazine (25 mg) in 2 ml 50% methanol/water wasadded to bovine serum albumin (30 mg) in 2 ml distilled water withstirring. The pH was adjusted to 5.5 with 0.1N HCl.1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (1 g) in 2 ml distilledwater was added in four parts adjusting the pH with 0.1N HCl after eachaddition to about pH 5.5. The mixture was then stirred at roomtemperature for 18 hours. The mixture was dialysed in a cellulosedialysing tube (Spectra/Por^(R), MW 12,000-14,000) against distilledwater for four days. The solution from the dialysing tube was found tocontain 2.7 mg/ml protein via the Biuret protein concentrationdetermining method.

EXAMPLE II 4-(1-(1-Phenylcyclohexyl)) piperidinyl ChloroformateImmunogen

4-(1-(-Phenylcyclohexyl)) piperidinyl chloroformate (57 mg) in 0.25 mldimethylformamide was added to bovine serum albumin (50 mg) in 2.5 ml0.1M phosphate buffer pH 8.0 and stirred at room temperature for onehour. The mixture was dialysed in a cellulose dialysing tube(Spectra/Por^(R), MW 12,000-14,000) against distilled water for 2 daysand 0.90% saline for one day. The solution from the dialysing tube wasfound to contain 15.4 mg/ml protein via the Biuret protein concentrationdetermining method.

EXAMPLE III 4-Amino-1-(1-phenylcyclohexyl) piperidine Immunogen

4-Amino-1-(1-phenylcyclohexyl) piperidine (40 mg) in 2.5 mldimethylformamide and 7.5 ml distilled water was added to bovine serumalbumin (69.5 mg) in 2.0 ml distilled water with stirring and the pH wasadjusted to pH 5.0-5.5 with 0.1N HCl.1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (60 mg) was added whilemaintaining the pH at 5.0-5.5 with 0.1N HCl. The mixture was stirred atroom temperature for 2 hours. The mixture was dialysed in a cellulosedialysing tube (Spectra/Por^(R), MW 12,000-14,000) against distilledwater for 3 days. The solution from the dialysing tube was found tocontain 4.19 mg/ml protein via the Biuret protein concentrationdetermining method.

EXAMPLE IV 1-Benzyl-4-(1-cyanocyclohexyl) piperazine

1-Benzylpiperazine (Aldrich) (6.12 g) was dissolved in 10 ml deionizedwater and cooled to 0° C. Concentrated hydrochloric acid (3.6 ml) wasadded to adjust the pH to 5. After warming to room temperature, 3.6 mlcyclohexanone was added, followed by potassium cyanide (2.4 g) in 6 mldeionized water. The solution slowly became cloudy. After 18 hours, thesolid was filtered. The solid was dissolved in chloroform, dried overNa₂ SO₄, filtered and the solvent was removed in vacuo to yield pure1-benzyl-4-(1-cyanocyclohexyl) piperazine.

EXAMPLE V 1-Benzyl-4-(1-phenylcyclohexyl) piperazine

To bromobenzene (0.6 g) and magnesium turnings (0.2 g) in 30 ml drytetrahydrofuran under nitrogen was added a small crystal of iodine and10 drops of 1,2-dibromoethane. After small bubbles began to form, thereaction was stirred and heated to reflux for 4 hours. After cooling toroom temperature, 1-benzyl-4-(1-cyanocyclohexyl)piperazine (1 g) wasadded. After 23 hours, the reaction was filtered, 20 ml saturatedaqueous ammonium chloride was added and the mixture was extracted withdiethyl ether. The ether was dried and removed in vacuo. The residue waschromatographed on silica gel eluted with chloroform to yield pure1-benzyl-4-(1-phenylcyclohexyl)piperazine.

EXAMPLE VI 1-(1-Phenylcyclohexyl)piperazine

1-Benzyl-4-(1-phenylcyclohexyl)piperazine (0.47 g) was dissolved in 86ml methanol and 14 ml 0.2N HCl in methanol. The mixture was hydrogenatedover palladium black (0.1175 g) and 3 atm. of hydrogen at roomtemperature for 1 hour. The reaction was filtered and the solvent wasremoved in vacuo. The residue was chromatographed on silica gel elutedwith the appropriate mixture of methanol and chloroform to yield pure1-(1-phenylcyclohexyl)piperazine.

EXAMPLE VII 1-(1-Cyanocyclohexyl)-4-hydroxyl piperidine

4-Hydroxypiperidine (Aldrich) (5 g) was dissolved in distilled water (14ml), cooled to 0° C. and the pH was adjusted to between 4 and 5 byaddition of concentrated hydrochloric acid and 4-hydroxypiperidine.After warming to room temperature, 5.2 ml cyclohexanone and 3.3 gpotassium cyanide in water (9 ml) were added sequentially. After 18hours stirring at room temperature, the solid was filtered. The solidwas dissolved in 100 ml methylene chloride, dried over Na₂ SO₄, filteredand the solvent was removed in vacuo to yield pure1-(1-cyanocyclohexyl)-4-hydroxypiperidine.

EXAMPLE VIII 1-(1-Phenylcyclohexyl)-4-hydroxypiperidine

To bromobenzene (4.1 ml) and magnesium turnings (4 g) in 200 ml drytetrahydrofuran under nitrogen was added a crystal of iodine and 10drops of 1,2-dibromoethane. After small bubbles began to form, thereaction was stirred and heated to reflux for 4 hours. After cooling toroom temperature 1-(1-cyanocyclohexyl)-4-hydroxypiperidine (2 g) wasadded. After 17 hours, the reaction was filtered, 120 ml saturatedammonium chloride was added and the mixture was extracted with ethylether. The ether layer was dried and the solvent was removed in vacuo.The residue was chromatographed on silica gel eluted with theappropriate mixture of methanol and chloroform to yield pure1-(1-phenylcyclohexyl)-4-hydroxypiperidine.

EXAMPLE IX 4-(1-(1-Phenylcyclohexyl))piperidinyl Chloroformate

1-(1-Phenylcyclohexyl)-4-hydroxypiperidine (0.261 mg) was suspended in 5ml dry benzene and 2 ml 10% phosgene in benzene was added. Afterstirring, stoppered for 3 hours at room temperature, 1 ml of chloroformwas added. After 30 minutes, the solvent was removed in vacuo. Carbontetrachloride (dry) (1 ml) was added and removed in vacuo to yield4-(1-(1-phenylcyclohexyl))piperidinyl chloroformate as a white solid.

EXAMPLE X 4-(5-Fluoresceinylcarbamoyl)-1-(phenylcyclohexyl) piperidine

4-(1-(1-Phenylcyclohexyl))piperidinyl chloroformate (10 mg) and5-aminofluorescein (10 mg) were dissolved in 2 ml dry pyridine andstirred at room temperature for about 18 hours. The reaction waschromatographed on silica gel preparative plates eluted with theappropriate ratio of methanol, chloroform and acetic acid to yield thedesired tracer.

EXAMPLE XI 1-(1-Phenylcyclohexyl)-4-piperidone

1-(1-Phenylcyclohexyl)-4-hydroxypiperidine was dissolved in 15 mlglacial acetic acid and 0.3 ml concentrated sulfuric acid, and 1.9 mlJones reagent (made from 26.72 g H₂ CrO₄ and 23 ml H₂ SO₄ diluted to 100ml with water) was added dropwise. After stirring at room temperaturefor 20 minutes, 2 ml 2-propanol, Zn(Hg) (made from 1.5 g mossy zinc and0.2 g mercuric chloride in 20 ml water and 0.25 ml concentratedhydrochloric acid at room temperature for 5 minutes), sodium citratedihydrate (3.6 g) and 35 ml deionized water were added sequentially.After stirring the solution at room temperature for 30 minutes, thereaction was extracted with chloroform. The solvent was removed invacuo. Distilled water was added and removed in vacuo. The residue waschromatographed on silica gel eluted with the appropriate mixture ofmethanol and chloroform to yield pure1-(1-phenylcyclohexyl)-4-piperidone.

Example XII 4-Amino-1-(1-phenylcyclohexyl)piperidine

1-(1-Phenylcyclohexyl)-4-piperidone (0.245 g) and ammonium acetate (1 g)were dissolved in 3.5 ml dry methanol. After stirring at roomtemperature for 10 minutes, sodium cyanoborohydride (0.1 g) was addedand the reaction stoppered. After 20 hours, 0.1 ml concentratedhydrochloric acid was added followed by 10 ml water and the mixture wasstirred for 1.5 hours. The solution was basified with potassiumcarbonate to pH 9 and extracted with methylene chloride. The methylenechloride was dried over Na₂ SO₄ and removed in vacuo. The productdistilled as a colorless, clear oil at 90°-130° C. in vacuo (about 5mmHg).

Example XIII 4-(4-Chloro-6-(5-fluoresceinylamino)-1,3,5-triazin-2-yl)amino-1-(1-phenylcyclohexyl)piperidine

4-Amino-1-(1-phenylcyclohexyl)piperidine (9 mg) and5-((4,6-dichloro-1,3,5-triazin-2-yl)amino) fluorescein (17 mg) weredissolved in 1 ml methanol and 0.1 ml triethylamine. After stirring atroom temperature for 30 hours, the solvent was removed in vacuo. Theresidue was chromatographed on silica gel preparative plates eluted withthe appropriate mixture of methanol and chloroform to yield the tracer.

Example XIV 4-(4-Chloro-6-(6-fluoresceinylamino)-1,3,5-triazin-2-yl)amino-1-(1-phenylcyclohexyl)piperidine

The same procedure was used as in Example XIII except that6-((4,6-dichloro-1,3,5-triazin-2-yl)amino) fluorescein was used insteadof 5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein.

Example XV 4-(Fluorescein-6-ylcarbonyl)amino-1-(1-phenylcyclohexyl)piperidine

6-Carboxyfluorescein (Calbiochem)(14 mg), N-hydroxysuccinimide (6 mg)and N,N'-dicyclohexylcarbodiimide (14 mg) were dissolved in 0.5 ml drypyridine and stirred at room temperature, stoppered. After 1 hour,4-amino-1-(1-phenylcyclohexyl) piperidine (9 mg) was added followed by0.5 ml dry pyridine. After 15 hours, the reaction was chromatographed onsilica gel preparative plates eluted with the appropriate mixture ofmethanol, chloroform and acetic acid.

Example XVI 4-(Fluorescein-5-ylcarbonyl)amino-1-(1-phenycyclohexyl)piperidine

The same procedure was used as in Example XV except 5-carboxyfluorescein(Calbiochem) was used instead of 6-carboxyfluorescein.

We claim:
 1. A process for detecting the presence of phencyclidine andphencyclidine metabolites which comprises the steps of:(a) contacting asample with riboflavin binding protein, with a phencyclidine derivativeantiserum, and with a compound capable of producing a detectablefluorescence polarization response to the presence of the phencyclidinederivative antiserum; wherein said compound has the structure: ##STR4##in which Q is fluorescein, or a fluorescein derivative,Z is NH, CO orCNH, n is O or 1 when W is N and 1 when W is CH, R is a linking groupincluding up to 4 heteroatoms and having a total of from 0 to 8 carbonatoms and heteroatoms, and W is CH or N, andwherein said phencyclidinederivative antiserum contains antibodies to a compound having thestructure ##STR5## in which Q is a poly(amino acid) or a poly(aminoacid) derivative, and Z, n, R and W are as defined above, (b) passingplane polarized light through the resulting solution from step (a) toobtain a fluorescence polarization response; and (c) detecting thefluorescence polarization response of the solution of step (b) as ameasure of the presence of phencyclidine and phencyclidine metabolitesin the sample.
 2. The process of claim 1 wherein Q of said compoundcapable of producing a detectable fluorescence polarization response tothe presence of the phencyclidine derivative antiserum is an amino, anamido, an amidino, a urea, a thiourea, a carbamido, a thiocarbamido, atriazinylamino, or a (carboxyamino)sulfonamido derivative offluorescein.
 3. The process of claim 1 wherein Q of said compoundcapable of producing a detectable fluorescence polarization response tothe presence of the phencyclidine derivative antiserum is4-chloro-6-(fluorescein-5-ylamino)-1,3,5-triazin-2-yl.
 4. The process ofclaim 1 wherein Q of said compound capable of producing a detectablefluorescence polarization response to the presence of the phencyclidinederivative antiserum is4-chloro-6-(fluorescein-6-ylamino)-1,3,5-triazin-2-yl.
 5. The process ofclaim 1 wherein Q of said compound capable of producing a detectablefluorescence polarization response to the presence of the phencyclidinederivative antiserum is fluorescein-5-ylcarbonyl.
 6. The process ofclaim 1 wherein Q of said compound capable of producing a detectablefluorescence polarization response to the presence of the phencyclidinederivative antiserum is fluorescein-6-ylcarbonyl.
 7. The process ofclaim 1 wherein Q of said compound capable of producing a detectablefluorescence polarization response to the presence of the phencyclidinederivative antiserum is (fluorescein-5-ylamino)carbonyl.
 8. The processof claim 1 wherein Q of said compound capable of producing a detectablefluorescence polarization response to the presence of the phencyclidinederivative antiserum is (fluorescein-6-ylamino)carbonyl.